4ebp 1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc 4ebp 1
    4ebp 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    4ebp 1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc 4ebp 1
    4ebp 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phospho 4ebp 1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho 4ebp 1
    Mesangial cells were transfected with miR-21 Sponge or vector. The serum-starved cells were incubated with 2 ng/ml TGFβ for 24 hours. The cell lysates were immunoblotted with phospho-S6 kinase (Thr-389), S6 kinase (panel A), phospho-mTOR (Ser-2448), mTOR (panel B), <t>phospho-4EBP-1</t> (Thr-34/46), phospho-4EBP-1 (Ser-65) and 4EBP-1 (panel C) antibodies as indicated.
    Phospho 4ebp 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "TGFβ-Stimulated MicroRNA-21 Utilizes PTEN to Orchestrate AKT/mTORC1 Signaling for Mesangial Cell Hypertrophy and Matrix Expansion"

    Article Title: TGFβ-Stimulated MicroRNA-21 Utilizes PTEN to Orchestrate AKT/mTORC1 Signaling for Mesangial Cell Hypertrophy and Matrix Expansion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042316

    Mesangial cells were transfected with miR-21 Sponge or vector. The serum-starved cells were incubated with 2 ng/ml TGFβ for 24 hours. The cell lysates were immunoblotted with phospho-S6 kinase (Thr-389), S6 kinase (panel A), phospho-mTOR (Ser-2448), mTOR (panel B), phospho-4EBP-1 (Thr-34/46), phospho-4EBP-1 (Ser-65) and 4EBP-1 (panel C) antibodies as indicated.
    Figure Legend Snippet: Mesangial cells were transfected with miR-21 Sponge or vector. The serum-starved cells were incubated with 2 ng/ml TGFβ for 24 hours. The cell lysates were immunoblotted with phospho-S6 kinase (Thr-389), S6 kinase (panel A), phospho-mTOR (Ser-2448), mTOR (panel B), phospho-4EBP-1 (Thr-34/46), phospho-4EBP-1 (Ser-65) and 4EBP-1 (panel C) antibodies as indicated.

    Techniques Used: Transfection, Plasmid Preparation, Incubation

    4e bp1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc 4e bp1
    TM6SF2 lacking promotes cell survival and carcinogenesis. ( A ) Cell growth was assessed through MTS assay for 24, 48, 72 hours and 1 week (λ = 490 nm). Data are expressed as fold increase vs control. ( B ) Representative images of wound healing assay were acquired at 0, 24, 48 hours (magnification, 100×). The dotted lines indicate the scratch width. ( C ) Phosphorylation of Akt at serine 437 residue (P[S437]-Akt), mTOR at serine 2448 residue (p[S2448]-mTOR), phosphorylation <t>of</t> <t>4E-BP1</t> at threonine 37/46 residues P[Thr37/46]-4E-BP1, and total Akt, mTOR, and 4E-BP1 were evaluated by Western blot. ( D–F ) Quantification of P(S437)-Akt/total Akt, p(S2448)-mTOR/total mTOR, and P(Thr37/46)-4E-BP1/total 4E-BP1 ratios were measured through ImageJ software and normalized to the β-actin housekeeping gene. At least 3 independent experiments were conducted. For bar graphs, data are expressed as means and SE. Adjusted ∗ P < .05 and ∗∗ P < .01 vs Cas9 + and/or vs MBOAT7 -/- . AU, arbitrary unit.
    4e Bp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "TM6SF2/PNPLA3/MBOAT7 Loss-of-Function Genetic Variants Impact on NAFLD Development and Progression Both in Patients and in In Vitro Models"

    Article Title: TM6SF2/PNPLA3/MBOAT7 Loss-of-Function Genetic Variants Impact on NAFLD Development and Progression Both in Patients and in In Vitro Models

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    doi: 10.1016/j.jcmgh.2021.11.007

    TM6SF2 lacking promotes cell survival and carcinogenesis. ( A ) Cell growth was assessed through MTS assay for 24, 48, 72 hours and 1 week (λ = 490 nm). Data are expressed as fold increase vs control. ( B ) Representative images of wound healing assay were acquired at 0, 24, 48 hours (magnification, 100×). The dotted lines indicate the scratch width. ( C ) Phosphorylation of Akt at serine 437 residue (P[S437]-Akt), mTOR at serine 2448 residue (p[S2448]-mTOR), phosphorylation of 4E-BP1 at threonine 37/46 residues P[Thr37/46]-4E-BP1, and total Akt, mTOR, and 4E-BP1 were evaluated by Western blot. ( D–F ) Quantification of P(S437)-Akt/total Akt, p(S2448)-mTOR/total mTOR, and P(Thr37/46)-4E-BP1/total 4E-BP1 ratios were measured through ImageJ software and normalized to the β-actin housekeeping gene. At least 3 independent experiments were conducted. For bar graphs, data are expressed as means and SE. Adjusted ∗ P < .05 and ∗∗ P < .01 vs Cas9 + and/or vs MBOAT7 -/- . AU, arbitrary unit.
    Figure Legend Snippet: TM6SF2 lacking promotes cell survival and carcinogenesis. ( A ) Cell growth was assessed through MTS assay for 24, 48, 72 hours and 1 week (λ = 490 nm). Data are expressed as fold increase vs control. ( B ) Representative images of wound healing assay were acquired at 0, 24, 48 hours (magnification, 100×). The dotted lines indicate the scratch width. ( C ) Phosphorylation of Akt at serine 437 residue (P[S437]-Akt), mTOR at serine 2448 residue (p[S2448]-mTOR), phosphorylation of 4E-BP1 at threonine 37/46 residues P[Thr37/46]-4E-BP1, and total Akt, mTOR, and 4E-BP1 were evaluated by Western blot. ( D–F ) Quantification of P(S437)-Akt/total Akt, p(S2448)-mTOR/total mTOR, and P(Thr37/46)-4E-BP1/total 4E-BP1 ratios were measured through ImageJ software and normalized to the β-actin housekeeping gene. At least 3 independent experiments were conducted. For bar graphs, data are expressed as means and SE. Adjusted ∗ P < .05 and ∗∗ P < .01 vs Cas9 + and/or vs MBOAT7 -/- . AU, arbitrary unit.

    Techniques Used: MTS Assay, Wound Healing Assay, Western Blot, Software

    List of Antibodies and Relative Dilutions Used in Western Blot and Immunocytochemistry Experiments
    Figure Legend Snippet: List of Antibodies and Relative Dilutions Used in Western Blot and Immunocytochemistry Experiments

    Techniques Used: Western Blot, Immunocytochemistry

    4e bp1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc 4e bp1
    4e Bp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti pi 4e bp1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti pi 4e bp1
    Everolimus inhibited proliferation and mTOR signaling in HCC cell lines. (a) Dose-dependent inhibition of HCC cell proliferation by everolimus. The effect of everolimus on cell viability was assessed by MTT assay. Dose-response curves of everolimus for all HCC cell lines were shown. Similar results were observed in 3 independent experiments. (b) Average IC 50 values of everolimus in HCC cell lines. Cumulative results from 3 independent experiments were shown as mean ± SEM. (c) Everolimus inhibited the mTOR pathway in HCC cells. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with 0.1 μ M everolimus (hereafter labeled as Eve) or DMSO control for 48 hrs and 72 hrs. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), <t>S6,</t> <t>pi-4E-BP1</t> <t>(ser65),</t> and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.
    Anti Pi 4e Bp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Enhanced Antitumor Activity with Combining Effect of mTOR Inhibition and Microtubule Stabilization in Hepatocellular Carcinoma"

    Article Title: Enhanced Antitumor Activity with Combining Effect of mTOR Inhibition and Microtubule Stabilization in Hepatocellular Carcinoma

    Journal: International Journal of Hepatology

    doi: 10.1155/2013/103830

    Everolimus inhibited proliferation and mTOR signaling in HCC cell lines. (a) Dose-dependent inhibition of HCC cell proliferation by everolimus. The effect of everolimus on cell viability was assessed by MTT assay. Dose-response curves of everolimus for all HCC cell lines were shown. Similar results were observed in 3 independent experiments. (b) Average IC 50 values of everolimus in HCC cell lines. Cumulative results from 3 independent experiments were shown as mean ± SEM. (c) Everolimus inhibited the mTOR pathway in HCC cells. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with 0.1 μ M everolimus (hereafter labeled as Eve) or DMSO control for 48 hrs and 72 hrs. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.
    Figure Legend Snippet: Everolimus inhibited proliferation and mTOR signaling in HCC cell lines. (a) Dose-dependent inhibition of HCC cell proliferation by everolimus. The effect of everolimus on cell viability was assessed by MTT assay. Dose-response curves of everolimus for all HCC cell lines were shown. Similar results were observed in 3 independent experiments. (b) Average IC 50 values of everolimus in HCC cell lines. Cumulative results from 3 independent experiments were shown as mean ± SEM. (c) Everolimus inhibited the mTOR pathway in HCC cells. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with 0.1 μ M everolimus (hereafter labeled as Eve) or DMSO control for 48 hrs and 72 hrs. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

    Techniques Used: Inhibition, MTT Assay, Labeling, Expressing, Western Blot

    Enhanced antitumor activity of the everolimus/patupilone combination in HCC cell lines. (a) Effects of everolimus/patupilone in HCC cell lines. HepG2, Hep3B, and SNU398 cells (1 × 10 4 ) were treated with various concentrations of everolimus in combination with 0.5 nM patupilone (Pat) for 24 hrs. Cell viability was assessed by MTT assay. Cumulative results from 3 independent experiments were shown as mean ± SEM (* P < 0.05, ** P < 0.01, *** P < 0.001 versus everolimus-treated group). (b) The mTOR signaling in HCC cells was not further suppressed by the everolimus/patupilone combination treatment. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with everolimus (0.1 μ M) and/or patupilone (Pat) (0.5 nM) for 24 hrs. The everolimus/patupilone combination is abbreviated as Eve/Pat hereafter. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.
    Figure Legend Snippet: Enhanced antitumor activity of the everolimus/patupilone combination in HCC cell lines. (a) Effects of everolimus/patupilone in HCC cell lines. HepG2, Hep3B, and SNU398 cells (1 × 10 4 ) were treated with various concentrations of everolimus in combination with 0.5 nM patupilone (Pat) for 24 hrs. Cell viability was assessed by MTT assay. Cumulative results from 3 independent experiments were shown as mean ± SEM (* P < 0.05, ** P < 0.01, *** P < 0.001 versus everolimus-treated group). (b) The mTOR signaling in HCC cells was not further suppressed by the everolimus/patupilone combination treatment. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with everolimus (0.1 μ M) and/or patupilone (Pat) (0.5 nM) for 24 hrs. The everolimus/patupilone combination is abbreviated as Eve/Pat hereafter. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

    Techniques Used: Activity Assay, MTT Assay, Expressing, Western Blot

    anti 4e bp1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti 4e bp1
    Everolimus inhibited proliferation and mTOR signaling in HCC cell lines. (a) Dose-dependent inhibition of HCC cell proliferation by everolimus. The effect of everolimus on cell viability was assessed by MTT assay. Dose-response curves of everolimus for all HCC cell lines were shown. Similar results were observed in 3 independent experiments. (b) Average IC 50 values of everolimus in HCC cell lines. Cumulative results from 3 independent experiments were shown as mean ± SEM. (c) Everolimus inhibited the mTOR pathway in HCC cells. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with 0.1 μ M everolimus (hereafter labeled as Eve) or DMSO control for 48 hrs and 72 hrs. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), <t>S6,</t> <t>pi-4E-BP1</t> (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.
    Anti 4e Bp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Enhanced Antitumor Activity with Combining Effect of mTOR Inhibition and Microtubule Stabilization in Hepatocellular Carcinoma"

    Article Title: Enhanced Antitumor Activity with Combining Effect of mTOR Inhibition and Microtubule Stabilization in Hepatocellular Carcinoma

    Journal: International Journal of Hepatology

    doi: 10.1155/2013/103830

    Everolimus inhibited proliferation and mTOR signaling in HCC cell lines. (a) Dose-dependent inhibition of HCC cell proliferation by everolimus. The effect of everolimus on cell viability was assessed by MTT assay. Dose-response curves of everolimus for all HCC cell lines were shown. Similar results were observed in 3 independent experiments. (b) Average IC 50 values of everolimus in HCC cell lines. Cumulative results from 3 independent experiments were shown as mean ± SEM. (c) Everolimus inhibited the mTOR pathway in HCC cells. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with 0.1 μ M everolimus (hereafter labeled as Eve) or DMSO control for 48 hrs and 72 hrs. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.
    Figure Legend Snippet: Everolimus inhibited proliferation and mTOR signaling in HCC cell lines. (a) Dose-dependent inhibition of HCC cell proliferation by everolimus. The effect of everolimus on cell viability was assessed by MTT assay. Dose-response curves of everolimus for all HCC cell lines were shown. Similar results were observed in 3 independent experiments. (b) Average IC 50 values of everolimus in HCC cell lines. Cumulative results from 3 independent experiments were shown as mean ± SEM. (c) Everolimus inhibited the mTOR pathway in HCC cells. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with 0.1 μ M everolimus (hereafter labeled as Eve) or DMSO control for 48 hrs and 72 hrs. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

    Techniques Used: Inhibition, MTT Assay, Labeling, Expressing, Western Blot

    Enhanced antitumor activity of the everolimus/patupilone combination in HCC cell lines. (a) Effects of everolimus/patupilone in HCC cell lines. HepG2, Hep3B, and SNU398 cells (1 × 10 4 ) were treated with various concentrations of everolimus in combination with 0.5 nM patupilone (Pat) for 24 hrs. Cell viability was assessed by MTT assay. Cumulative results from 3 independent experiments were shown as mean ± SEM (* P < 0.05, ** P < 0.01, *** P < 0.001 versus everolimus-treated group). (b) The mTOR signaling in HCC cells was not further suppressed by the everolimus/patupilone combination treatment. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with everolimus (0.1 μ M) and/or patupilone (Pat) (0.5 nM) for 24 hrs. The everolimus/patupilone combination is abbreviated as Eve/Pat hereafter. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.
    Figure Legend Snippet: Enhanced antitumor activity of the everolimus/patupilone combination in HCC cell lines. (a) Effects of everolimus/patupilone in HCC cell lines. HepG2, Hep3B, and SNU398 cells (1 × 10 4 ) were treated with various concentrations of everolimus in combination with 0.5 nM patupilone (Pat) for 24 hrs. Cell viability was assessed by MTT assay. Cumulative results from 3 independent experiments were shown as mean ± SEM (* P < 0.05, ** P < 0.01, *** P < 0.001 versus everolimus-treated group). (b) The mTOR signaling in HCC cells was not further suppressed by the everolimus/patupilone combination treatment. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with everolimus (0.1 μ M) and/or patupilone (Pat) (0.5 nM) for 24 hrs. The everolimus/patupilone combination is abbreviated as Eve/Pat hereafter. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

    Techniques Used: Activity Assay, MTT Assay, Expressing, Western Blot

    p 4ebp1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p 4ebp1
    P 4ebp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti phospho 4e bp1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho 4e bp1
    A. SUM-149 and MCF10A cells were treated with vehicle (0mg/mL, SV or MV) or 0.5mg/mL Reishi (SR or MR) for 24 hours before lysis. Western blot analyses were completed for total eIF4G, eIF4A, eIF4E, <t>and</t> <t>4E-BP1</t> obtained from m7GTP pull-down lysates and whole cell lysates. B. Graph represents quantification of eIF4F complex as in Dumstorf et al., 2010 , where eIF4G normalized to eIF4E is divided by 4E-BP1 normalized to eIF4E [(eIF4G/eIF4E)/(4E-BP1/eIF4E)]. Number of biological replicates (n) varies among experiments (SUM-149; n = 3, MCF10A; n = 1). Columns show means ± SEM of integrated density units, shown relative to vehicle controls. Reishi significantly reduces eIF4F complex assembly at * P <0.02 in IBC SUM-149 cells. C. 1×10 5 cells (SUM-149 and MCF10A) were seeded per well in a six well plate and treated with vehicle or 0.5 mg/mL Reishi for 24 hours. The treatment was removed and the cells were re-incubated for 30 minutes in methionine/cysteine - free DMEM. L-[ 35 S] Methionine and L-[ 35 S] Cysteine (2 mCi/mL) +2% FBS was then added to the cultures. Total cell lysates prepared in NP-40 lysis buffer were analyzed for incorporated radioactivity in trichloroacetic acid precipitates. Data are expressed as means ± SEM of duplicate determinations. Experiment was repeated three times. Reishi significantly (* P <0.05) reduces protein synthesis by 48%.
    Anti Phospho 4e Bp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Anti-Tumor Effects of Ganoderma lucidum (Reishi) in Inflammatory Breast Cancer in In Vivo and In Vitro Models"

    Article Title: Anti-Tumor Effects of Ganoderma lucidum (Reishi) in Inflammatory Breast Cancer in In Vivo and In Vitro Models

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0057431

    A. SUM-149 and MCF10A cells were treated with vehicle (0mg/mL, SV or MV) or 0.5mg/mL Reishi (SR or MR) for 24 hours before lysis. Western blot analyses were completed for total eIF4G, eIF4A, eIF4E, and 4E-BP1 obtained from m7GTP pull-down lysates and whole cell lysates. B. Graph represents quantification of eIF4F complex as in Dumstorf et al., 2010 , where eIF4G normalized to eIF4E is divided by 4E-BP1 normalized to eIF4E [(eIF4G/eIF4E)/(4E-BP1/eIF4E)]. Number of biological replicates (n) varies among experiments (SUM-149; n = 3, MCF10A; n = 1). Columns show means ± SEM of integrated density units, shown relative to vehicle controls. Reishi significantly reduces eIF4F complex assembly at * P <0.02 in IBC SUM-149 cells. C. 1×10 5 cells (SUM-149 and MCF10A) were seeded per well in a six well plate and treated with vehicle or 0.5 mg/mL Reishi for 24 hours. The treatment was removed and the cells were re-incubated for 30 minutes in methionine/cysteine - free DMEM. L-[ 35 S] Methionine and L-[ 35 S] Cysteine (2 mCi/mL) +2% FBS was then added to the cultures. Total cell lysates prepared in NP-40 lysis buffer were analyzed for incorporated radioactivity in trichloroacetic acid precipitates. Data are expressed as means ± SEM of duplicate determinations. Experiment was repeated three times. Reishi significantly (* P <0.05) reduces protein synthesis by 48%.
    Figure Legend Snippet: A. SUM-149 and MCF10A cells were treated with vehicle (0mg/mL, SV or MV) or 0.5mg/mL Reishi (SR or MR) for 24 hours before lysis. Western blot analyses were completed for total eIF4G, eIF4A, eIF4E, and 4E-BP1 obtained from m7GTP pull-down lysates and whole cell lysates. B. Graph represents quantification of eIF4F complex as in Dumstorf et al., 2010 , where eIF4G normalized to eIF4E is divided by 4E-BP1 normalized to eIF4E [(eIF4G/eIF4E)/(4E-BP1/eIF4E)]. Number of biological replicates (n) varies among experiments (SUM-149; n = 3, MCF10A; n = 1). Columns show means ± SEM of integrated density units, shown relative to vehicle controls. Reishi significantly reduces eIF4F complex assembly at * P <0.02 in IBC SUM-149 cells. C. 1×10 5 cells (SUM-149 and MCF10A) were seeded per well in a six well plate and treated with vehicle or 0.5 mg/mL Reishi for 24 hours. The treatment was removed and the cells were re-incubated for 30 minutes in methionine/cysteine - free DMEM. L-[ 35 S] Methionine and L-[ 35 S] Cysteine (2 mCi/mL) +2% FBS was then added to the cultures. Total cell lysates prepared in NP-40 lysis buffer were analyzed for incorporated radioactivity in trichloroacetic acid precipitates. Data are expressed as means ± SEM of duplicate determinations. Experiment was repeated three times. Reishi significantly (* P <0.05) reduces protein synthesis by 48%.

    Techniques Used: Lysis, Western Blot, Incubation, Radioactivity

    4ebp1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc 4ebp1
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    A. SUM-149 and MCF10A cells were treated with vehicle (0mg/mL, SV or MV) or 0.5mg/mL Reishi (SR or MR) for 24 hours before lysis. Western blot analyses were completed for total eIF4G, eIF4A, eIF4E, <t>and</t> <t>4E-BP1</t> obtained from m7GTP pull-down lysates and whole cell lysates. B. Graph represents quantification of eIF4F complex as in Dumstorf et al., 2010 , where eIF4G normalized to eIF4E is divided by 4E-BP1 normalized to eIF4E [(eIF4G/eIF4E)/(4E-BP1/eIF4E)]. Number of biological replicates (n) varies among experiments (SUM-149; n = 3, MCF10A; n = 1). Columns show means ± SEM of integrated density units, shown relative to vehicle controls. Reishi significantly reduces eIF4F complex assembly at * P <0.02 in IBC SUM-149 cells. C. 1×10 5 cells (SUM-149 and MCF10A) were seeded per well in a six well plate and treated with vehicle or 0.5 mg/mL Reishi for 24 hours. The treatment was removed and the cells were re-incubated for 30 minutes in methionine/cysteine - free DMEM. L-[ 35 S] Methionine and L-[ 35 S] Cysteine (2 mCi/mL) +2% FBS was then added to the cultures. Total cell lysates prepared in NP-40 lysis buffer were analyzed for incorporated radioactivity in trichloroacetic acid precipitates. Data are expressed as means ± SEM of duplicate determinations. Experiment was repeated three times. Reishi significantly (* P <0.05) reduces protein synthesis by 48%.
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    A. SUM-149 and MCF10A cells were treated with vehicle (0mg/mL, SV or MV) or 0.5mg/mL Reishi (SR or MR) for 24 hours before lysis. Western blot analyses were completed for total eIF4G, eIF4A, eIF4E, <t>and</t> <t>4E-BP1</t> obtained from m7GTP pull-down lysates and whole cell lysates. B. Graph represents quantification of eIF4F complex as in Dumstorf et al., 2010 , where eIF4G normalized to eIF4E is divided by 4E-BP1 normalized to eIF4E [(eIF4G/eIF4E)/(4E-BP1/eIF4E)]. Number of biological replicates (n) varies among experiments (SUM-149; n = 3, MCF10A; n = 1). Columns show means ± SEM of integrated density units, shown relative to vehicle controls. Reishi significantly reduces eIF4F complex assembly at * P <0.02 in IBC SUM-149 cells. C. 1×10 5 cells (SUM-149 and MCF10A) were seeded per well in a six well plate and treated with vehicle or 0.5 mg/mL Reishi for 24 hours. The treatment was removed and the cells were re-incubated for 30 minutes in methionine/cysteine - free DMEM. L-[ 35 S] Methionine and L-[ 35 S] Cysteine (2 mCi/mL) +2% FBS was then added to the cultures. Total cell lysates prepared in NP-40 lysis buffer were analyzed for incorporated radioactivity in trichloroacetic acid precipitates. Data are expressed as means ± SEM of duplicate determinations. Experiment was repeated three times. Reishi significantly (* P <0.05) reduces protein synthesis by 48%.
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    Mesangial cells were transfected with miR-21 Sponge or vector. The serum-starved cells were incubated with 2 ng/ml TGFβ for 24 hours. The cell lysates were immunoblotted with phospho-S6 kinase (Thr-389), S6 kinase (panel A), phospho-mTOR (Ser-2448), mTOR (panel B), phospho-4EBP-1 (Thr-34/46), phospho-4EBP-1 (Ser-65) and 4EBP-1 (panel C) antibodies as indicated.

    Journal: PLoS ONE

    Article Title: TGFβ-Stimulated MicroRNA-21 Utilizes PTEN to Orchestrate AKT/mTORC1 Signaling for Mesangial Cell Hypertrophy and Matrix Expansion

    doi: 10.1371/journal.pone.0042316

    Figure Lengend Snippet: Mesangial cells were transfected with miR-21 Sponge or vector. The serum-starved cells were incubated with 2 ng/ml TGFβ for 24 hours. The cell lysates were immunoblotted with phospho-S6 kinase (Thr-389), S6 kinase (panel A), phospho-mTOR (Ser-2448), mTOR (panel B), phospho-4EBP-1 (Thr-34/46), phospho-4EBP-1 (Ser-65) and 4EBP-1 (panel C) antibodies as indicated.

    Article Snippet: Phospho-Akt (Ser473), phospho-Akt (thr-308), Akt, phospho-S6 kinase, S6 kinase, phospho-4EBP-1 (Thr-37/46) phospho-4EBP-1 (Ser-65) 4EBP-1, phospho-GSK3β, GSK3β, phospho-tuberin (Thr 1462), tuberin, phospho-PRAS40 (Thr 246), PRAS40, phospho-mTOR (Ser-2448) and mTOR antibodies were purchased from Cell Signaling, Boston, MA. siRNA pool of three oligonucleotides against PTEN mRNA, collagen II (α2) and PTEN antibodies were obtained from Santacruz, Delaware, CA.

    Techniques: Transfection, Plasmid Preparation, Incubation

    TM6SF2 lacking promotes cell survival and carcinogenesis. ( A ) Cell growth was assessed through MTS assay for 24, 48, 72 hours and 1 week (λ = 490 nm). Data are expressed as fold increase vs control. ( B ) Representative images of wound healing assay were acquired at 0, 24, 48 hours (magnification, 100×). The dotted lines indicate the scratch width. ( C ) Phosphorylation of Akt at serine 437 residue (P[S437]-Akt), mTOR at serine 2448 residue (p[S2448]-mTOR), phosphorylation of 4E-BP1 at threonine 37/46 residues P[Thr37/46]-4E-BP1, and total Akt, mTOR, and 4E-BP1 were evaluated by Western blot. ( D–F ) Quantification of P(S437)-Akt/total Akt, p(S2448)-mTOR/total mTOR, and P(Thr37/46)-4E-BP1/total 4E-BP1 ratios were measured through ImageJ software and normalized to the β-actin housekeeping gene. At least 3 independent experiments were conducted. For bar graphs, data are expressed as means and SE. Adjusted ∗ P < .05 and ∗∗ P < .01 vs Cas9 + and/or vs MBOAT7 -/- . AU, arbitrary unit.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: TM6SF2/PNPLA3/MBOAT7 Loss-of-Function Genetic Variants Impact on NAFLD Development and Progression Both in Patients and in In Vitro Models

    doi: 10.1016/j.jcmgh.2021.11.007

    Figure Lengend Snippet: TM6SF2 lacking promotes cell survival and carcinogenesis. ( A ) Cell growth was assessed through MTS assay for 24, 48, 72 hours and 1 week (λ = 490 nm). Data are expressed as fold increase vs control. ( B ) Representative images of wound healing assay were acquired at 0, 24, 48 hours (magnification, 100×). The dotted lines indicate the scratch width. ( C ) Phosphorylation of Akt at serine 437 residue (P[S437]-Akt), mTOR at serine 2448 residue (p[S2448]-mTOR), phosphorylation of 4E-BP1 at threonine 37/46 residues P[Thr37/46]-4E-BP1, and total Akt, mTOR, and 4E-BP1 were evaluated by Western blot. ( D–F ) Quantification of P(S437)-Akt/total Akt, p(S2448)-mTOR/total mTOR, and P(Thr37/46)-4E-BP1/total 4E-BP1 ratios were measured through ImageJ software and normalized to the β-actin housekeeping gene. At least 3 independent experiments were conducted. For bar graphs, data are expressed as means and SE. Adjusted ∗ P < .05 and ∗∗ P < .01 vs Cas9 + and/or vs MBOAT7 -/- . AU, arbitrary unit.

    Article Snippet: Anti–P-Ser473 Akt, Akt, P(Thr37/46)-4E-BP1, 4E-BP1, P(Ser2448)-mTOR, and mTOR antibodies were acquired from Cell Signaling Technology (Danvers, MA).

    Techniques: MTS Assay, Wound Healing Assay, Western Blot, Software

    List of Antibodies and Relative Dilutions Used in Western Blot and Immunocytochemistry Experiments

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: TM6SF2/PNPLA3/MBOAT7 Loss-of-Function Genetic Variants Impact on NAFLD Development and Progression Both in Patients and in In Vitro Models

    doi: 10.1016/j.jcmgh.2021.11.007

    Figure Lengend Snippet: List of Antibodies and Relative Dilutions Used in Western Blot and Immunocytochemistry Experiments

    Article Snippet: Anti–P-Ser473 Akt, Akt, P(Thr37/46)-4E-BP1, 4E-BP1, P(Ser2448)-mTOR, and mTOR antibodies were acquired from Cell Signaling Technology (Danvers, MA).

    Techniques: Western Blot, Immunocytochemistry

    Everolimus inhibited proliferation and mTOR signaling in HCC cell lines. (a) Dose-dependent inhibition of HCC cell proliferation by everolimus. The effect of everolimus on cell viability was assessed by MTT assay. Dose-response curves of everolimus for all HCC cell lines were shown. Similar results were observed in 3 independent experiments. (b) Average IC 50 values of everolimus in HCC cell lines. Cumulative results from 3 independent experiments were shown as mean ± SEM. (c) Everolimus inhibited the mTOR pathway in HCC cells. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with 0.1 μ M everolimus (hereafter labeled as Eve) or DMSO control for 48 hrs and 72 hrs. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

    Journal: International Journal of Hepatology

    Article Title: Enhanced Antitumor Activity with Combining Effect of mTOR Inhibition and Microtubule Stabilization in Hepatocellular Carcinoma

    doi: 10.1155/2013/103830

    Figure Lengend Snippet: Everolimus inhibited proliferation and mTOR signaling in HCC cell lines. (a) Dose-dependent inhibition of HCC cell proliferation by everolimus. The effect of everolimus on cell viability was assessed by MTT assay. Dose-response curves of everolimus for all HCC cell lines were shown. Similar results were observed in 3 independent experiments. (b) Average IC 50 values of everolimus in HCC cell lines. Cumulative results from 3 independent experiments were shown as mean ± SEM. (c) Everolimus inhibited the mTOR pathway in HCC cells. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with 0.1 μ M everolimus (hereafter labeled as Eve) or DMSO control for 48 hrs and 72 hrs. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

    Article Snippet: The following antibodies were used in the study: anti-mTOR, anti-pi-mTOR (ser2448), anti-Akt, anti-pi-Akt (ser473), anti-p70S6k, anti-pi-p70S6k (Thr389), anti-S6, anti-pi-S6 (ser240/244), anti-4E-BP1, anti-pi-4E-BP1 (ser65), anti-cleaved PARP (all from Cell Signaling Technology, Beverly, MA, USA), and anti-actin (Calbiochem, Nottingham, UK).

    Techniques: Inhibition, MTT Assay, Labeling, Expressing, Western Blot

    Enhanced antitumor activity of the everolimus/patupilone combination in HCC cell lines. (a) Effects of everolimus/patupilone in HCC cell lines. HepG2, Hep3B, and SNU398 cells (1 × 10 4 ) were treated with various concentrations of everolimus in combination with 0.5 nM patupilone (Pat) for 24 hrs. Cell viability was assessed by MTT assay. Cumulative results from 3 independent experiments were shown as mean ± SEM (* P < 0.05, ** P < 0.01, *** P < 0.001 versus everolimus-treated group). (b) The mTOR signaling in HCC cells was not further suppressed by the everolimus/patupilone combination treatment. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with everolimus (0.1 μ M) and/or patupilone (Pat) (0.5 nM) for 24 hrs. The everolimus/patupilone combination is abbreviated as Eve/Pat hereafter. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

    Journal: International Journal of Hepatology

    Article Title: Enhanced Antitumor Activity with Combining Effect of mTOR Inhibition and Microtubule Stabilization in Hepatocellular Carcinoma

    doi: 10.1155/2013/103830

    Figure Lengend Snippet: Enhanced antitumor activity of the everolimus/patupilone combination in HCC cell lines. (a) Effects of everolimus/patupilone in HCC cell lines. HepG2, Hep3B, and SNU398 cells (1 × 10 4 ) were treated with various concentrations of everolimus in combination with 0.5 nM patupilone (Pat) for 24 hrs. Cell viability was assessed by MTT assay. Cumulative results from 3 independent experiments were shown as mean ± SEM (* P < 0.05, ** P < 0.01, *** P < 0.001 versus everolimus-treated group). (b) The mTOR signaling in HCC cells was not further suppressed by the everolimus/patupilone combination treatment. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with everolimus (0.1 μ M) and/or patupilone (Pat) (0.5 nM) for 24 hrs. The everolimus/patupilone combination is abbreviated as Eve/Pat hereafter. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

    Article Snippet: The following antibodies were used in the study: anti-mTOR, anti-pi-mTOR (ser2448), anti-Akt, anti-pi-Akt (ser473), anti-p70S6k, anti-pi-p70S6k (Thr389), anti-S6, anti-pi-S6 (ser240/244), anti-4E-BP1, anti-pi-4E-BP1 (ser65), anti-cleaved PARP (all from Cell Signaling Technology, Beverly, MA, USA), and anti-actin (Calbiochem, Nottingham, UK).

    Techniques: Activity Assay, MTT Assay, Expressing, Western Blot

    Everolimus inhibited proliferation and mTOR signaling in HCC cell lines. (a) Dose-dependent inhibition of HCC cell proliferation by everolimus. The effect of everolimus on cell viability was assessed by MTT assay. Dose-response curves of everolimus for all HCC cell lines were shown. Similar results were observed in 3 independent experiments. (b) Average IC 50 values of everolimus in HCC cell lines. Cumulative results from 3 independent experiments were shown as mean ± SEM. (c) Everolimus inhibited the mTOR pathway in HCC cells. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with 0.1 μ M everolimus (hereafter labeled as Eve) or DMSO control for 48 hrs and 72 hrs. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

    Journal: International Journal of Hepatology

    Article Title: Enhanced Antitumor Activity with Combining Effect of mTOR Inhibition and Microtubule Stabilization in Hepatocellular Carcinoma

    doi: 10.1155/2013/103830

    Figure Lengend Snippet: Everolimus inhibited proliferation and mTOR signaling in HCC cell lines. (a) Dose-dependent inhibition of HCC cell proliferation by everolimus. The effect of everolimus on cell viability was assessed by MTT assay. Dose-response curves of everolimus for all HCC cell lines were shown. Similar results were observed in 3 independent experiments. (b) Average IC 50 values of everolimus in HCC cell lines. Cumulative results from 3 independent experiments were shown as mean ± SEM. (c) Everolimus inhibited the mTOR pathway in HCC cells. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with 0.1 μ M everolimus (hereafter labeled as Eve) or DMSO control for 48 hrs and 72 hrs. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

    Article Snippet: The following antibodies were used in the study: anti-mTOR, anti-pi-mTOR (ser2448), anti-Akt, anti-pi-Akt (ser473), anti-p70S6k, anti-pi-p70S6k (Thr389), anti-S6, anti-pi-S6 (ser240/244), anti-4E-BP1, anti-pi-4E-BP1 (ser65), anti-cleaved PARP (all from Cell Signaling Technology, Beverly, MA, USA), and anti-actin (Calbiochem, Nottingham, UK).

    Techniques: Inhibition, MTT Assay, Labeling, Expressing, Western Blot

    Enhanced antitumor activity of the everolimus/patupilone combination in HCC cell lines. (a) Effects of everolimus/patupilone in HCC cell lines. HepG2, Hep3B, and SNU398 cells (1 × 10 4 ) were treated with various concentrations of everolimus in combination with 0.5 nM patupilone (Pat) for 24 hrs. Cell viability was assessed by MTT assay. Cumulative results from 3 independent experiments were shown as mean ± SEM (* P < 0.05, ** P < 0.01, *** P < 0.001 versus everolimus-treated group). (b) The mTOR signaling in HCC cells was not further suppressed by the everolimus/patupilone combination treatment. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with everolimus (0.1 μ M) and/or patupilone (Pat) (0.5 nM) for 24 hrs. The everolimus/patupilone combination is abbreviated as Eve/Pat hereafter. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

    Journal: International Journal of Hepatology

    Article Title: Enhanced Antitumor Activity with Combining Effect of mTOR Inhibition and Microtubule Stabilization in Hepatocellular Carcinoma

    doi: 10.1155/2013/103830

    Figure Lengend Snippet: Enhanced antitumor activity of the everolimus/patupilone combination in HCC cell lines. (a) Effects of everolimus/patupilone in HCC cell lines. HepG2, Hep3B, and SNU398 cells (1 × 10 4 ) were treated with various concentrations of everolimus in combination with 0.5 nM patupilone (Pat) for 24 hrs. Cell viability was assessed by MTT assay. Cumulative results from 3 independent experiments were shown as mean ± SEM (* P < 0.05, ** P < 0.01, *** P < 0.001 versus everolimus-treated group). (b) The mTOR signaling in HCC cells was not further suppressed by the everolimus/patupilone combination treatment. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with everolimus (0.1 μ M) and/or patupilone (Pat) (0.5 nM) for 24 hrs. The everolimus/patupilone combination is abbreviated as Eve/Pat hereafter. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

    Article Snippet: The following antibodies were used in the study: anti-mTOR, anti-pi-mTOR (ser2448), anti-Akt, anti-pi-Akt (ser473), anti-p70S6k, anti-pi-p70S6k (Thr389), anti-S6, anti-pi-S6 (ser240/244), anti-4E-BP1, anti-pi-4E-BP1 (ser65), anti-cleaved PARP (all from Cell Signaling Technology, Beverly, MA, USA), and anti-actin (Calbiochem, Nottingham, UK).

    Techniques: Activity Assay, MTT Assay, Expressing, Western Blot

    A. SUM-149 and MCF10A cells were treated with vehicle (0mg/mL, SV or MV) or 0.5mg/mL Reishi (SR or MR) for 24 hours before lysis. Western blot analyses were completed for total eIF4G, eIF4A, eIF4E, and 4E-BP1 obtained from m7GTP pull-down lysates and whole cell lysates. B. Graph represents quantification of eIF4F complex as in Dumstorf et al., 2010 , where eIF4G normalized to eIF4E is divided by 4E-BP1 normalized to eIF4E [(eIF4G/eIF4E)/(4E-BP1/eIF4E)]. Number of biological replicates (n) varies among experiments (SUM-149; n = 3, MCF10A; n = 1). Columns show means ± SEM of integrated density units, shown relative to vehicle controls. Reishi significantly reduces eIF4F complex assembly at * P <0.02 in IBC SUM-149 cells. C. 1×10 5 cells (SUM-149 and MCF10A) were seeded per well in a six well plate and treated with vehicle or 0.5 mg/mL Reishi for 24 hours. The treatment was removed and the cells were re-incubated for 30 minutes in methionine/cysteine - free DMEM. L-[ 35 S] Methionine and L-[ 35 S] Cysteine (2 mCi/mL) +2% FBS was then added to the cultures. Total cell lysates prepared in NP-40 lysis buffer were analyzed for incorporated radioactivity in trichloroacetic acid precipitates. Data are expressed as means ± SEM of duplicate determinations. Experiment was repeated three times. Reishi significantly (* P <0.05) reduces protein synthesis by 48%.

    Journal: PLoS ONE

    Article Title: Anti-Tumor Effects of Ganoderma lucidum (Reishi) in Inflammatory Breast Cancer in In Vivo and In Vitro Models

    doi: 10.1371/journal.pone.0057431

    Figure Lengend Snippet: A. SUM-149 and MCF10A cells were treated with vehicle (0mg/mL, SV or MV) or 0.5mg/mL Reishi (SR or MR) for 24 hours before lysis. Western blot analyses were completed for total eIF4G, eIF4A, eIF4E, and 4E-BP1 obtained from m7GTP pull-down lysates and whole cell lysates. B. Graph represents quantification of eIF4F complex as in Dumstorf et al., 2010 , where eIF4G normalized to eIF4E is divided by 4E-BP1 normalized to eIF4E [(eIF4G/eIF4E)/(4E-BP1/eIF4E)]. Number of biological replicates (n) varies among experiments (SUM-149; n = 3, MCF10A; n = 1). Columns show means ± SEM of integrated density units, shown relative to vehicle controls. Reishi significantly reduces eIF4F complex assembly at * P <0.02 in IBC SUM-149 cells. C. 1×10 5 cells (SUM-149 and MCF10A) were seeded per well in a six well plate and treated with vehicle or 0.5 mg/mL Reishi for 24 hours. The treatment was removed and the cells were re-incubated for 30 minutes in methionine/cysteine - free DMEM. L-[ 35 S] Methionine and L-[ 35 S] Cysteine (2 mCi/mL) +2% FBS was then added to the cultures. Total cell lysates prepared in NP-40 lysis buffer were analyzed for incorporated radioactivity in trichloroacetic acid precipitates. Data are expressed as means ± SEM of duplicate determinations. Experiment was repeated three times. Reishi significantly (* P <0.05) reduces protein synthesis by 48%.

    Article Snippet: We used a monoclonal anti-Phospho-mTOR (Ser2481), monoclonal anti-mTOR, anti-Phospho-4E-BP1 (Thr37/46), anti-Phospho-S6 Ribosomal Protein (Ser235/236), anti-S6 Ribosomal Protein, anti-Akt, anti-Phospho-Akt (Ser473), anti-eIF4G and anti-eIF4E and polyclonal anti-p70s6 kinase (Cell Signaling, Danvers, MA) antibodies.

    Techniques: Lysis, Western Blot, Incubation, Radioactivity